Cerebral Aβ deposition precedes reduced cerebrospinal fluid and serum Aβ42/Aβ40 ratios in the AppNL−F/NL−F knock-in mouse model of Alzheimer’s disease

Background Aβ42/Aβ40 ratios in cerebrospinal fluid (CSF) and blood are reduced in preclinical Alzheimer’s disease (AD), but their temporal and correlative relationship with cerebral Aβ pathology at this early disease stage is not well understood. In the present study, we aim to investigate such relationships using App knock-in mouse models of preclinical AD. Methods CSF, serum, and brain tissue were collected from 3- to 18-month-old AppNL−F/NL−F knock-in mice (n = 48) and 2–18-month-old AppNL/NL knock-in mice (n = 35). The concentrations of Aβ42 and Aβ40 in CSF and serum were measured using Single molecule array (Simoa) immunoassays. Cerebral Aβ plaque burden was assessed in brain tissue sections by immunohistochemistry and thioflavin S staining. Furthermore, the concentrations of Aβ42 in soluble and insoluble fractions prepared from cortical tissue homogenates were measured using an electrochemiluminescence immunoassay. Results In AppNL−F/NL−F knock-in mice, Aβ42/Aβ40 ratios in CSF and serum were significantly reduced from 12 and 16 months of age, respectively. The initial reduction of these biomarkers coincided with cerebral Aβ pathology, in which a more widespread Aβ plaque burden and increased levels of Aβ42 in the brain were observed from approximately 12 months of age. Accordingly, in the whole study population, Aβ42/Aβ40 ratios in CSF and serum showed a negative hyperbolic association with cerebral Aβ plaque burden as well as the levels of both soluble and insoluble Aβ42 in the brain. These associations tended to be stronger for the measures in CSF compared with serum. In contrast, no alterations in the investigated fluid biomarkers or apparent cerebral Aβ plaque pathology were found in AppNL/NL knock-in mice during the observation time. Conclusions Our findings suggest a temporal sequence of events in AppNL−F/NL−F knock-in mice, in which initial deposition of Aβ aggregates in the brain is followed by a decline of the Aβ42/Aβ40 ratio in CSF and serum once the cerebral Aβ pathology becomes significant. Our results also indicate that the investigated biomarkers were somewhat more strongly associated with measures of cerebral Aβ pathology when assessed in CSF compared with serum. Supplementary Information The online version contains supplementary material available at 10.1186/s13195-023-01196-8.


Animals
In addition to App NL-F/NL-F and App NL/NL knock-in mice, male and female 3xTg mice (3-24 months, n = 42) were used for assessment of Ab in CSF and brain tissue sections. Together with endogenous expression of M146V-mutated human presenilin 1 (PS1), these mice overexpress human APP with the Swedish (KM670/671NL) mutation as well as human MAPT(4R0N) with the P301L mutation under the control of the mouse Thy1 promoter element.
This results in cerebral accumulation of intracellular Ab from 3 months of age and deposition of extracellular amyloid plaques from 6 months of age (1), although phenotypic drift in the development of Ab pathology have been reported in various colonies (2).
Following overnight incubation, the sections were washed 3 x 10 minutes in TBSX, incubated with appropriate Alexa-fluorophore-conjugated secondary antibody diluted 1:200 in TBSX containing 2.5% NDS, washed 3 x 10 minutes in TBSX, mounted on glass slides and coverslipped with ProLong TM Diamond Antifade Mountant according to the recommendations from the manufacturer.

Image acquisition and analysis
Fluorescence images of whole brain sections from 3xTg mice were acquired using a 10x objective lens on the Operetta® CLS TM High Content Analysis System (PerkinElmer). Cortex from one brain section per mouse were manually segmented and the area covered by Aβpositive staining was quantified using the Fiji software by applying an automated local threshold that was maintained for all images analyzed. Figure S1: Experimental design The illustration shows the experimental design of the study (created with BioRender.com). Abbreviations: Ab, amyloid beta; APP, amyloid precursor protein; CSF, cerebrospinal fluid; ECL, electrochemiluminescence; FA, formic acid; Simoa, single molecule array; TBS, trisbuffered saline. Figure S2: CSF and serum Ab42 and Ab40 in App NL-F/NL-F knock-in mice CSF and serum Ab42 and Ab40 were measured in 3 (n = 9)-, 6 (n = 7)-, 9 (n = 8)-, 12 (n = 7)-, 16 (n = 8)-, and 18 (n = 9)-months-old App NL-F/NL-F knock-in mice. (A) CSF Ab42 showed a steady significant reduction from 12 months of age while (B) no age-dependent effect on CSF Ab40 was found. (C) Serum Ab42 was significantly reduced from 16 months of age while (D) serum Ab40 was significantly increased from this time point. (E) Serum Ab42 correlated significantly positive with CSF Ab42 while (F) no correlation between serum and CSF Ab40 was found. Data is presented as median and IQR. Whiskers represent data within 1.5IQR of the lower and upper quartiles. For comparison between groups, statistical analyses were perform using the Kruskal-Wallis H test followed by the Mann-Whitney U test for post hoc group comparisons (*p < 0.05, **p < 0.01, ***p < 0.001). Correlation analyses were performed using Spearman's rank-ordered correlation coefficient. Abbreviations: Ab, amyloid beta; CSF, cerebrospinal fluid; IQR, interquartile range.

Figure S3
: CSF and serum Ab42/Ab40 ratio, Ab42, and Ab40 in App NL/NL knock-in mice CSF and serum Ab42 and Ab40 were measured in 2 (n = 5)-, 4 (n = 6)-, 6 (n = 10)-, 9 (n = 9)-, and 18 (n = 5)-months-old App NL/NL knock-in mice. (A) Some fluctuations in the CSF Ab42/Ab40 ratio was found with age although no age-dependent effect on (B) CSF Ab42 or (C) CSF Ab40 was observed. No change in (D) the Ab42/Ab40 ratio, (E) Ab42, or (F) Ab40 in serum was found with age. There was no correlation between (G) the Ab42/Ab40 ratio, (H) Ab42, or (I) Ab40 in serum and corresponding measures in CSF. (J) App NL/NL knock-in mice did not show any apparent cerebral Ab deposition with age. Data is presented as median and IQR. Whiskers represent data within 1.5IQR of the lower and upper quartiles. For comparison between groups, statistical analyses were perform using the Kruskal-Wallis H test followed by the Mann-Whitney U test for post hoc group comparisons (*p < 0.05, **p < 0.01). Correlation analyses were performed using Spearman's rank-ordered correlation coefficient. Scale bar: 500 µm. Abbreviations: Ab, amyloid beta; CSF, cerebrospinal fluid; IQR, interquartile range.    Data is presented as median and IQR. For comparison between groups, statistical analyses were perform using the Kruskal-Wallis test followed by the Mann-Whitney U test for post hoc group comparisons. Abbreviations: Ab, amyloid beta; CSF, cerebrospinal fluid. a p < 0.01 vs. 3 months, b p < 0.001 vs. 3 months, c p < 0.05 vs. 6 months, d p < 0.01 vs. 6 months, e p < 0.001 vs. 6 months, f p < 0.05 vs. 9 months, g p < 0.001 vs. 9 months, h p < 0.01 vs. 12 months, i p < 0.001 vs. 12 months, j p < 0.05 vs. 16 months, k < 0.01 vs. 16 months, l < 0.001 vs. 16 months  Correlation analyses were performed using Spearman's rank-ordered correlation coefficient. Differences between correlation coefficients were estimated using Mengs Z-test. Abbreviations: Ab, amyloid beta; CSF, cerebrospinal fluid.